EA036409B1 - Phosphoramidate nucleoside derivatives as anticancer agents - Google Patents

Abstract

This invention relates to derivatives of cladribine of Formula (II). The compounds are phosphoramidate derivatives in which the phosphoramidate moiety is situated on the 3'-hydroxyl group of cladribine. The invention also relates to pharmaceutical formulations of the cladribine derivatives. The compounds are useful in the treatment of cancer.

Description

The present invention relates to cladribine derivatives. The compounds are phosphoramidate derivatives in which the phosphoramidate moiety is located at the 3'-hydroxyl group of cladribine. The present invention also relates to pharmaceutical formulations of cladribine derivatives. The compounds are useful in the treatment of cancer.

State of the art

Several modified purine nucleosides are known to exhibit potent biological properties, including chemotherapeutic potential. One example is cladribine 1, a useful but toxic drug. It is administered by infusion for the treatment of leukemia, and in particular hairy cell leukemia. It is also used for chronic lymphocytic leukemia in patients who do not respond to standard regimens with alkylating agents.

As with all nucleoside analogs, these agents require intracellular kinase-mediated activation of their bioactive 5'-phosphate forms.

WO 2006100439 describes certain phosphoramidate derivatives of cladribine and their anti-cancer activities. The phosphoramidates described in WO 2006100439 are located on the 5'-hydroxyl group of cladribine, for example compound Y, and the phosphoramidate moiety acts as a prodrug for the nucleoside monophosphate.

OH OH

Cladribine Y.

Phosphoramidates such as Compound Y are known as ProTides. ProTides may provide significant benefits by improving the anti-cancer properties of nucleosides or increasing potency, or by avoiding mechanisms and intrinsic and acquired resistance ('Application of ProTide

Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development '; Slusarczyk et al; J. Med. Chem .; 2014, 57, 1531-1542; Phosphoramidate ProTides of the anticancer agent FUDR successfully deliver the preformed bioactive monophosphate in cells and confer advantage over the parent nucleoside; J.

Med. Chem .; 2011, 54, 7247-7258; and Vande Voorde et al .; The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5fluoro-2'-deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes; Biochem. Pharmacol .; 2011, 82, 441-452).

However, while 5'-phosphoramidates have shown excellent anti-cancer activity, this is usually not the case for 3'-phosphoramidates, which usually have only poor anti-cancer activity.

An object of some embodiments of the present invention is to provide novel anti-cancer compounds. It is an object of some embodiments of the present invention to provide compounds that are more potent anti-cancer compounds than prior art compounds.

An object of some embodiments of the present invention is to provide novel compounds that target cancer stem cells. An object of some embodiments of the present invention is to provide compounds that are more effective at targeting cancer stem cells than prior art compounds.

An object of some embodiments of the present invention is to provide compounds that are less susceptible to mycoplasma infection than prior art compounds.

It is an object of some embodiments of the present invention to provide compounds that are less affected by cancer resistance mechanisms than prior art compounds.

Certain embodiments of the present invention achieve some or all of the above objectives.

Brief description of the invention

According to the present invention, a compound of formula (II) or a pharmaceutically acceptable salt thereof is provided:

(Ii)

R ¹ is phenyl or naphthyl;

R ^{2 is} selected from C ^ C ^ -alkyl, C ₃ -C7 -cycloalkyl or O-alkylene-phenyl;

each R ³ and R ^{4 is} independently selected from H, O-Cb-alkyl and O-C ^ alklen-K ⁹ ;

R ⁹ is phenyl;

where any phenyl, alkyl, alkylene, cycloalkyl or naphthyl group is optionally substituted with 1-4 substituents selected from halogen, nitro, cyano, NR ^a R ^a , NR ^a S (O) 2R ^a , NR ^a C (O) R ^a , NR ^a CONR ^a R ^a , NR ^a CO2R ^a , OR ^a ; SR ^a , SOR ^a , SO3R ^a , SO2R ^a , SO2NR ^a R ^a , CO2R ^a C (O) R ^a , CONR ^a R ^a , CR ^a R ^a NR ^a R ^a , С1-С ₄ -alkyl, С ₂ - C ₄ alkenyl, C ₂ -C ₄ alkynyl and C ₁ -C ₄ haloalkyl;

where R ^{a is} independently selected from H and C ₁ -C ₄ -alkyl.

Surprisingly, the 3'-cladribine phosphoramidates of the present invention were more effective than the corresponding 5'-cladribine ProTides. This is contrary to the trend observed in general for other nucleosides.

Certain 3'-cladribine phosphoramidates of the present invention have been shown to target cancer stem cells.

The activity of the 3'-cladribine phosphoramidates of the present invention in cells infected with mycoplasma was less reduced than for cladribine and 5'-cladribine ProTides.

The following statements apply to compounds of formula (II). These statements are independent and interchangeable. In other words, any of the features described in any of the following statements may (where chemically acceptable) be combined with the features described in one or more of the other statements below. In particular, when a compound is exemplified or illustrated herein, any two or more statements below that describe a feature of a given compound, expressed at any level of generalization, may be combined to represent the subject matter that is intended to form part of the description of the present invention. in this invention.

It is possible that R ¹ is phenyl. It is possible that R ¹ is naphthyl (eg 1-naphthyl).

Preferably, R ¹ can be unsubstituted phenyl or unsubstituted naphthyl (eg 1-naphthyl). Thus, R ¹ can be unsubstituted phenyl. Alternatively, R ¹ can be unsubstituted naphthyl (eg, 1-naphthyl). R ¹ can be tetrahydronaphthyl.

R ² can be selected from C ₂ -C ₁₀ alkyl, C ₃ -C ₇ cycloalkyl, or C ₁ alkylene phenyl.

R ^{2 is} preferably selected so that it contains five or more carbon atoms. Therefore, R ² can be selected to contain six or more carbon atoms. R ^{2 is} preferably selected to contain only carbon and hydrogen atoms. R ² can be selected from C5-C7 cycloalkyl, C ₅ -C ₈ -alkyl and benzyl.

R ² can be C ² -C ¹⁰ alkyl. R ² can be C ₄ -C ₈ alkyl. Thus, R ² can be selected from isobutyl, tert-butyl, n-butyl, n-pentyl, CH ₂ C (Me) _3, or n-hexyl.

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R ² can be C ₃ -C ₇ cycloalkyl. Thus, R ² can be cyclohexyl.

R ² can be Cj alkylene phenyl. R ² can be benzyl.

It is possible that one of R ³ and R ⁴ is H and the other is selected so that it is a side chain of an amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, histidine, serine, cysteine, glutamic acid , aspartic acid, asparagine, glutamine, arginine, lysine, threonine, tyrosine and tryptophan. It is possible that one of R ³ and R ⁴ is H and the other is selected to be a side chain of an amino acid selected from glycine, alanine, valine, leucine, isoleucine and phenylalanine. It is possible that one R ³ and R ⁴ is H and the other is selected to be a side chain of an amino acid selected from alanine, valine, leucine, isoleucine and phenylalanine.

It is possible that R ⁴ is H and R ^{3 is} selected to be a side chain of an amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, histidine, serine, cysteine, glutamic acid, aspartic acid, asparagine, glutamine, arginine, lysine, threonine, tyrosine and tryptophan. It is possible that R ⁴ is H and R ^{3 is} selected to be a side chain of an amino acid selected from glycine, alanine, valine, leucine, isoleucine, and phenylalanine. It is possible that R ⁴ is H and R ^{3 is} selected to be a side chain of an amino acid selected from alanine, valine, leucine, isoleucine, and phenylalanine. Thus, the amino acid (NH2CR ³ R ⁴ CO2H) from which the phosphoramidate moiety is derived may be an L-amino acid.

It is possible that R ⁴ is H. It is possible that R ^{3 is} selected from H, C -C ^ alkyl, and C1-C3 alkylene-R ⁹ . It is possible that R ^{3 is} selected from C-C ^ alkyl and C ₁ -C ₃ -alkylene-And ⁹ . It is possible that R ⁹ is phenyl.

It is possible that one of R and R ⁴ is H and the other is selected from H, Me, isopropyl, isobutyl and benzyl. It is possible that one of R ³ and R ⁴ is H and the other is selected from Me, isopropyl, isobutyl and benzyl. It is possible that one of R ³ and R ⁴ is H and the other is Me.

It is possible that R ⁴ is H and R ^{3 is} selected from H, Me, isopropyl, isobutyl and benzyl. It is possible that R ⁴ is H and R ^{3 is} selected from Me, isopropyl, isobutyl and benzyl. It is possible that R ⁴ is H and R ³ is Me.

It is possible that R ³ is C ₁ -C ₄ -alkyl. It is possible that R ^{3 is} selected from isopropyl, isobutyl and methyl. It is possible that R ³ is CH 2 phenyl.

It is possible that R ³ is H. It is possible that R ^{4 is} selected from H, C -C ^ alkyl and C1-C3 alkylene-R ⁹ It is possible that R ^{4 is} selected from ^ -C ^ alkyl and C ₁ -C ₃ -alkylene -And ⁹ . It is possible that R ⁹ is phenyl.

It is possible that R ⁴ is C1-C4 alkyl. It is possible that R ^{4 is} selected from isopropyl, isobutyl and methyl. It is possible that R ⁴ is CH 2 phenyl.

It is possible that each R ⁴ and R ³ is methyl.

It is possible that R ⁴ is H, R ³ is Me and R ² is benzyl.

The compound of formula (II) can be a compound selected from

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2-chloro-2'-deoxyadenosine-3'- [phenyl (ethoxy-L-alanyl)] phosphate,

2-chloro-2'-deoxyadenosine-3'- [phenyl (tert-butoxy-Lalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-Dalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxyglycinyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-Lleucinyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl- (2,2-dimethylpropoxyL-alanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl (pentoxy-Lleucinyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl (cyclohexoxy-Lalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (cyclohexoxy-Lalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (2,2-dimethylpropoxy-balanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (ethoxy-2,2dimethylglycinyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-Lphenylalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl- (benzoxy-Lphenylalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-Lvalinyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (isopropoxy-Lalanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl (2-butoxy-L-alanyl)] phosphate,

2-Chloro-2'-deoxyadenosine-3'- [phenyl ((S) -1-phenylethoxy-balanyl) phosphate,

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl (benzoxy-Lalanyl) phosphate and

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-b-alanyl) phosphate.

The compound of formula (II) can be a compound selected from:

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl (benzoxy-Lalanyl) phosphate and

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-b-alanyl) phosphate.

It is possible that the compound of formula (II) is a free base.

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The compounds of the present invention contain a chiral center at the phosphorus atom. The compounds may be present as a mixture of phosphate diastereoisomers, as a (b) -epimer at phosphorus in substantially diastereomerically pure form, or as an (R) -epimer at phosphorus in substantially diastereomerically pure form. Substantially diastereomerically pure is defined for the purposes of the present invention as greater than about 90% diastereomeric purity. If present in a substantially diastereomerically pure form, the compound may have a diastereomeric purity of greater than 95, 98, 99, or even 99.5%. Alternatively, the compound may be present as a mixture of phosphate diastereoisomers.

The (R) - and / or (b) -epimers of the compounds can be obtained in substantially diastereomerically pure form by chromatography, for example HPLC, optionally using a chiral column. Alternatively, the (R) - and / or (b) -epimers of the compounds can be obtained in a substantially diastereomerically pure form by crystallization from a suitable solvent or solvent system. Alternatively, the (R) - and / or (b) -epimers of the compounds can be prepared in substantially diastereomerically pure form by condensing a suitably protected cladribine derivative with a diastereomerically enriched phosphoramidate precursor and then deprotecting. The (R) - and / or (b) -epimers of the compounds can be obtained in substantially diastereomerically pure form by direct synthesis, for example using the methods described in WO 2014/076490.

In a second aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising a therapeutically effective amount of a compound of formula (II) and a pharmaceutically acceptable excipient.

Various aspects of the present invention are based on the discovery that the compound of the present invention is capable of preferentially reducing the number of cancer stem cells. This finding is unexpected because cancer stem cells are known to be resistant to many chemotherapeutic agents and there has been no previous suggestion that the compound of the present invention or cladribine, the parent compound from which the compound of the present invention is prepared, is capable of targeting cancer stem cells. ... Thus, the discovery that a compound of the present invention is capable of targeting cancer stem cells and thus reducing their numbers, a finding that the inventors of the present invention have confirmed is useful for a wide variety of cancers, represents a surprising breakthrough that provides a kit new therapeutic uses for the compound of the present invention.

Detailed description

The compounds in the compositions of the present invention can be prepared, stored and / or administered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, wine, maleic, hydroxymalic, fumaric, apple, lemon, milk, mucous, gluconic, benzoic, amber, oxalic, phenylacetic, methanesulfo, toluenesulfo, benzenesulfo, salicylic, sulfanilic, aspartic, glutamic, oleanic, stealic pantothenic, digallic, ascorbic and valeric acids. Suitable base salts are prepared from bases which form non-toxic salts. Examples include salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc. Hemisalts of acids and bases can also be prepared, for example, hemisulfate, hemioxalate and hemicalcium salts. Preferably, the compounds of the present invention are not in salt form, i. E. they are in the free base / free acid form.

The term Cm-Cn refers to a group of m-n carbon atoms.

The term alkyl refers to a linear or branched saturated hydrocarbon group. The alkyl group is monovalent. For example, G-Cb-alkyl can refer to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl. Alkyl groups are preferably unsubstituted.

The term cycloalkyl refers to a cyclic saturated hydrocarbon group. The alkyl group is monovalent. For example, C ₅ -C ₇ -cycloalkyl can refer to cyclopentyl, cyclohexyl, or cycloheptyl. Cycloalkyl groups are preferably unsubstituted.

The term alkylene refers to a linear saturated hydrocarbon chain. The alkylene group is divalent. For example, Ci-alkylene can refer to a CH ₂ group. C ₂ -alkylene may refer to the —CH ₂ CH ₂ - group. Alkylene groups are preferably unsubstituted.

The term alkenyl refers to a branched or straight hydrocarbon chain containing at least one carbon-carbon double bond. Double bond (s) can be represented as the E or Z isomer. The double bond can be at any position in the hydrocarbon chain. For example, C ₂ -C ₄ alkenyl can refer to ethenyl, allyl and butenyl. Alkenyl groups are preferably 5,036409 unsubstituted.

The term alkynyl refers to a branched or straight hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond can be at any position in the hydrocarbon chain. For example, a C2-C ₆ -alkynyl can refer to ethynyl, propynyl, butynyl. Alkynyl groups are preferably unsubstituted.

The term aryl refers to phenyl groups, naphthyl groups, and tetrahydronaphthyl groups. The term aryl can refer to phenyl groups or naphthyl groups. Aryl groups (eg, naphthyl or phenyl groups) may be unsubstituted.

The present invention also includes all pharmaceutically acceptable isotopically labeled forms of compounds wherein one or more atoms have been replaced with atoms having the same atomic number, but an atomic mass or mass number other than the atomic mass or mass number of the predominant isotope commonly found in nature.

Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen such as 2H and ³ H, carbon such as C. ¹³ C and ¹⁴ C, chlorine such as ³⁶ Cl, fluorine such as ¹⁸ F, iodine, such as ¹²³ I and ¹²⁵ I, nitrogen such as ¹³ N and ¹⁵ N, oxygen such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus such as ³² P, and sulfur such as ³⁵ S.

Certain isotopically labeled compounds, for example, radioactive isotope introducing compounds, are useful in drug and / or substrate tissue distribution studies. Radioactive isotopes of tritium, i.e. ³ H, carbon-14, i.e. ¹⁴ C and ¹⁸ F are particularly useful for this purpose in view of their ease of administration and available detection methods.

Substitution with heavier isotopes such as deuterium, i.e. ² H may confer certain therapeutic benefits resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosages required, and therefore may be preferred in some circumstances.

Isotopically labeled compounds are generally prepared by conventional methods known to one of ordinary skill in the art, or by methods analogous to those described, using a suitable isotopically labeled reagent in place of the previously used unlabeled reagent.

The compounds of the present invention can be used in the treatment of the human body. They can be used to treat the body of an animal. In particular, the compounds of the present invention can be used to treat farm animals such as livestock. Alternatively, the compounds of the present invention can be used to treat companion animals such as cats, dogs, etc.

The compounds of the present invention may exist as a single crystalline form, or as a mixture of crystalline forms, or they may be amorphous. Thus, the compounds of the present invention contemplated for pharmaceutical use may be administered as crystalline or amorphous products. They can be obtained, for example, in the form of solid compacts, powders or films by methods such as precipitation, crystallization, lyophilization, or spray drying, or evaporation drying. Microwave or radio frequency drying can be used for this purpose.

For the above compounds of the present invention, the dose administered will, of course, vary depending on the compound employed, the route of administration, the treatment desired and the disorder indicated. For example, if the compound of the present invention is administered parenterally, the dose of the compound of the present invention may be in the range of 0.1 to 5 g / m ² , for example, 0.5 to 2 g / m ² . The dosage size for therapeutic purposes of the compounds of the present invention will generally vary depending on the nature and severity of the disease, the age and sex of the animal or patient, and the route of administration according to well-known medical principles.

Dose levels, dosing frequency and duration of treatment with compounds of the present invention are contemplated to vary depending on the composition and clinical indication, age and concomitant medical indication of the patient.

A compound of the present invention, or a pharmaceutically acceptable salt thereof, can be used as is, but will usually be administered as a pharmaceutical composition in which the compounds of the present invention or a pharmaceutically acceptable salt thereof are combined with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional methods for selecting and preparing suitable pharmaceutical formulations are described, for example, in Pharmaceuticals-The Science of Dosage Form Designs, M.E. Aulton, Churchill Livingstone, 1988.

Depending on the route of administration of the compounds of the present invention, the pharmaceutical composition that is used to administer the compounds of the present invention will preferably contain from 0.05 to 99% by weight of the compounds of the present invention, more preferably from 0.05 to 80% by weight of the compounds of the present invention, even more preferably from 0.10 to 70% by weight of the compound of the present invention, and even more preferably from 0.10 to 50% by weight of the compound of the present invention, all percentages by weight being based on the total composition.

For oral administration, the compounds of the present invention can be admixed with an adjuvant or carrier, eg lactose, sucrose, sorbitol, mannitol; starch such as potato starch, corn starch or amylopectin; cellulose derivatives; a binder such as gelatin or polyvinylpyrrolidone; and / or a lubricating agent such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin and the like, and then compressed into tablets. If coated tablets are required, the cores prepared as described above may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatin, talc and titanium oxide. Alternatively, the tablets can be coated with a suitable polymer dissolved in a readily volatile organic solvent.

To obtain soft gelatin capsules, the compounds of the present invention can be mixed with, for example, vegetable oil or polyethylene glycol. Hard gelatin capsules may contain granules of the compound using any of the above tablet auxiliaries. Also, liquid or semi-solid formulations of the compound of the present invention can be filled into hard gelatin capsules.

Liquid preparations for oral administration may be in the form of syrups or suspensions, for example solutions, containing a compound of the present invention, the remainder being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally, these liquid preparations may contain colorants, flavors, sweeteners (such as saccharin), preservatives, and / or carboxymethyl cellulose as a thickening agent, or other adjuvants known to a person skilled in the art.

For parenteral (eg, intravenous) administration, the compounds of the present invention may be administered as sterile aqueous or oily solutions. The compounds of the present invention are highly lipophilic. Therefore, the aqueous formulations can also contain a pharmaceutically acceptable polar organic solvent.

The dosage size for therapeutic purposes of the compounds of the present invention will generally vary depending on the nature and severity of the disease, the age and sex of the animal or patient, and the route of administration according to well known principles of medicine.

Dose levels, dosing frequency and duration of treatment with compounds of the present invention are contemplated to vary depending on the composition and clinical indication, age and concomitant medical indication of the patient.

Thus, the pharmaceutical compositions of the present invention may contain another active agent.

Cancer stem cells

Cancer stem cells, sometimes referred to as tumor initiator cells, are well known to those of ordinary skill in the art. As used in the present invention, the term cancer stem cell should be interpreted according to its generally accepted meaning, which is a cell that has the ability to self-renew through asymmetric division, inducing tumor formation and giving rise to more mature progeny of non-stem cancer cells by differentiation.

Cancer stem cells play an important role in the development, progression, recurrence and spread of cancer.

Accordingly, the discovery that the compounds of the present invention are capable of targeting cancer stem cells and thereby reducing their number provides a therapeutic opportunity for preventing or treating these activities.

As discussed in more detail elsewhere in this description, cancer stem cells are found in precancerous conditions, where their presence is believed to contribute to the development of these conditions to cancer. Accordingly, the methods of therapeutic and medical uses of the present invention, in which a compound of the present invention is used to target cancer stem cells, can be used to reduce the number of cancer stem cells in precancerous conditions (such as myelodysplastic syndrome or other conditions discussed elsewhere in this description ) and thus prevent the development of these precancerous conditions to cancer.

As referenced above, asymmetric cell division of cancer stem cells leads to differentiated non-stem cancer cells. Thus, cancer stem cells are responsible for the formation and maintenance of tumor volume.

The accumulation of these non-stem cancer cells plays an important role in the development of cancer. Targeting cancer stem cells with a compound of the present invention is capable of reducing the number of cancer stem cells, which in turn reduces the number of progeny non-stem cancer cells. Thus, the methods of therapeutic and medical uses of the compounds of the present invention according to the present invention are useful for treating cancer by preventing cancer progression. These embodiments are described in more detail elsewhere in this specification.

Cancer stem cells are also able to act as a reservoir of cancer cells, which can cause cancer to recur after remission. Even when most of the patient's cancer cells are

- 7,036409 leno (for example, surgery, radiotherapy or chemotherapy, either alone or in combination) so that no observable signs of cancer remain, the continued presence of cancer stem cells can cause cancer recurrence over time. Targeting cancer stem cells with a compound of the present invention provides a novel method that can reduce the number of cancer stem cells and kill cancer stem cells. Accordingly, and as discussed in more detail elsewhere herein, in suitable embodiments, the present invention relates to methods and medical uses in which a compound of the present invention prevents or delays cancer recurrence.

Moreover, the migration of cancer stem cells from the site of cancer to another location in the body can facilitate the spread of cancer, for example, leading to metastases. As a consequence, the ability of the compound of the present invention to target cancer stem cells, therefore, provides new therapeutic and medical uses in preventing or treating the spread of cancer.

In addition to their biological activities, cancer stem cells can be identified by their expression of certain characteristic cell surface markers. Cancer stem cells found in hematological malignancies are usually CD34, while in solid tumors CD44, CD133 and CD90 are identified as cancer stem cell markers. The following table lists examples of known surface phenotypes of cancer stem cells. It is contemplated that each of these forms of cancer stem cells can be targeted using a compound of the present invention according to the present invention, and that the methods or uses using the compound of the present invention can be used in the prevention or treatment of cancer associated with cancer stem cells expressing any from a given set of markers.

Tumor type Published cellular surface markers for cancer stem cells Solid tumors Breast CD4 4 + / CD2 4 ^- / ^low / Lineage ^- / ESA + CNS CD133 + Colon CD133 + Colon ESA ^hi 9 ^h / CD44 + / Lineage ^- / (CD166 +) Ewing CD133 + head and neck CD44 + / Lineage ^- Melanoma ABCB5 + Liver CD90 + / CD45- / (CD44 +) Cholangiocarcinoma CD44 + / GLI1 + (glioma-related oncogenic homolog1) Ovaries CD44 + / CD117 + Pancreas CD44 + / CD24 + / ESA + Pancreas CD133 + Non-small cell cancer lung CD44 + / Ber-EP4 + Bladder cancer CD4 4 + / ALDH1A1 +

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Hematologic tumors Acute myelocytic leukemia Lin ~ / CD34 ⁺ / CD38 ~ / CD123 ⁺ B-Acute lymphoblastic leukemia CD34 + / CD10 ^- or CD34 + / CD19 ^- B-Acute lymphoblastic leukemia CD34 + / CD38- / CD19 + Multiple myeloma CD34 "/ CD138" T-Acute lymphoblastic leukemia CD34 + / CD4 ^- or CD34 + / CD7 ^-

The data presented in the examples demonstrate that the compound of the present invention is capable of targeting cancer stem cells of leukemic stem cell lines, especially cancer stem cells present in the acute myelocytic leukemia KG1a cell line. This cell line displays a small stem cell-like compartment with excellent immunophenotype (Lin7CD34 ⁺ / CD387CD123 ⁺ ), which is targeted with the compound of the present invention. Accordingly, the methods of therapeutic or medical uses of the compounds of the present invention according to the present invention can be used to prevent or treat leukemia or other cancers associated with cancer stem cells expressing these characteristic markers.

The present invention also relates to methods and medical uses in which patients are selected to prevent or treat cancer using a compound of the present invention based on the identification of the presence of cancer stem cells in a biological sample representing a cancer or precancerous condition in the patient. The markers above provide suitable examples that can be used to identify the presence of cancer stem cells according to these embodiments of the present invention. Suitable methods by which you can examine the expression of these markers in a biological sample are considered additionally elsewhere in the present description.

Targeting cancer stem cells

The present invention relates to the first feature that the compounds of the present invention can be used to target cancer stem cells. The ability of the compounds of the present invention to target cancer stem cells is illustrated in the examples described in the present invention.

From the examples, it can be seen that when the compound of the present invention is provided to populations of cancer cells containing cancer stem cells, it targets the cancer stem cells present, resulting in a decrease in the total number of cancer cells and the proportion of total cancer cells possessing phenotypic markers of cancer stem cells. ...

While the parent prodrug cladribine is capable of targeting cancer stem cells at certain higher concentrations, the compounds of the present invention exhibit the ability to achieve this targeting over a wider concentration range. In particular, in vitro studies, the results of which are reported in this application, demonstrate that the compounds of the present invention are able to target cancer stem cells at low concentrations more effectively than cladribine. At certain concentrations, the improvement in targeting cancer stem cells is such that the proportion of cancer stem cells remaining in the population of cells treated with a compound of the present invention is significantly less than in the population of cells treated with cladribine. It is clear that the ability to achieve effective targeting of cancer stem cells at low concentrations of cytotoxic agents will generally be desirable as this reduces the likelihood of unwanted side effects.

It is believed that the compounds of the present invention have an increased permeability across the cell membrane (compared to cladribine) and that this contributes to the increased anticancer activity of the compounds of the present invention compared to the parent nucleoside from which they are obtained.

Without wishing to be bound by any hypothesis, the inventors believe that the decline in the number of cancer stem cells results from the targeted destruction of cancer stem cells in the cancer cell population. In other words, the compounds of the present invention appear to preferentially kill cancer stem cells as compared to killing non-stem cancer cells, thereby causing cancer stem cell death, and reducing the incidence of cancer.

- 9 036409 stem cells in the total population of cancer cells.

While the inventors believe that the compounds of the present invention preferentially kill cancer stem cells over non-stem cancer cells, other mechanisms may also contribute to a decrease in the cancer stem cell population caused by targeting these cells by a compound of the present invention.

Just by way of example, treatment with a compound of the present invention can cause increased differentiation of cancer stem cells, thereby reducing the number of cancer stem cells and also the proportion of total cancer cells represented by cancer stem cells. Alternatively, the compound of the present invention can cause cancer stem cells to lose their stem cell phenotype, for example, to lose their ability to replicate themselves, thereby decreasing the number of cancer stem cells.

References to targeting cancer stem cells throughout this specification should be interpreted accordingly. For the purposes of the present disclosure, cancer stem cell targeting can be used as including any mechanism by which a compound of the present invention reduces the proportion of cancer stem cells present in a cell population, either in vitro or in vivo. In particular, targeting cancer stem cells can be used as including preferentially killing cancer stem cells over other types of cells, in particular over non-stem cancer cells.

Preventing or treating cancer

The present invention relates to medical uses and methods of treatment in which a compound of the present invention is used to prevent or treat cancer. In the context of the present invention, the prevention of cancer should be considered to refer to the prophylactic uses of the compound of the present invention, used before the development of cancer, and in order to prevent the development of cancer. On the other hand, cancer treatment is applied as referring to the use of a compound of the present invention after the onset of cancer for the purpose of reducing the intensity of cancer by slowing or stopping the proliferation of cancer cells and tumor growth. Advantageously, cancer treatment can cause partial or complete reduction in the number of cancer cells and tumor size. An effective cancer treatment can cause the disease to either stabilize or respond to treatment according to the RECIST guidelines (criteria for evaluating the response of solid tumors).

As described in more detail below, the prevention of cancer according to the present invention may be particularly beneficial for patients who have a precancerous condition that increases their likelihood of developing cancer.

Cancer Prevention

Cancer prevention according to the present invention can be accomplished by treating a precancerous condition using a compound of the present invention according to various aspects or embodiments of the present invention described in the present invention.

In particular, cancer prevention in the context of the present invention can be accomplished by the methods or medical uses of the present invention in which a compound of the present invention is provided to a patient with a precancerous condition. The methods of therapeutic or medical uses according to this embodiment can prevent the development of a precancerous condition being treated prior to cancer, thereby providing effective cancer prevention.

References to cancer prevention in the context of the present invention may also include other prophylactic uses of a compound of the present invention. For example, the ability of a compound of the present invention to target cancer stem cells and thereby prevent cancer development and / or prevent cancer progression and / or prevent cancer recurrence and / or prevent cancer spread.

Precancerous conditions

Cancer is often preceded by the development of a precancerous condition that is not itself cancerous but is associated with an increased risk of cancer. The accumulation of genetic or epigenetic changes can induce the development of previously normal cells to the phenotype of cancer stem cells. Accordingly, cancer stem cells can also be present in these precancerous conditions as well as in cancerous conditions.

It is assumed that the presence of cancer stem cells in precancerous conditions promotes the development of these conditions before cancer. The methods and medical uses of the present invention can be used to target cancer stem cells present in precancerous conditions, and thereby treat these conditions. Clearly, the new and unexpected finding that the compounds of the present invention target cancer stem cells means that treatment of precancerous conditions with these compounds can be used to prevent the conditions being treated from developing to cancer. This is a method in which the compound of the present invention can be used medically in the prevention of cancer, as discussed elsewhere in the present description.

- 10 036409

Examples of precancerous conditions that can be treated according to the present invention include, but are not limited to, conditions selected from the group consisting of atrophic gastritis, congenital dyskeratosis, sideropenic dysphagia, lichen planus, oral submucosal fibrosis, solar elastosis, cervical dysplasia, leukoplakia, erythroplakia, monoclonal gammopathy of unclear significance (MGUS), monoclonal B-cell lymphocytosis (MBL), myelodysplastic syndromes, and precancerous gastric conditions such as atrophic gastritis, gastric ulcer, pernicious anemia, gastric stumps, gastric polyps, and gastric polyps. Among the precancerous gastric conditions listed, atrophic gastritis, pernicious anemia, gastric stumps and certain types of gastric polyps may particularly increase the risk of developing cancer.

Precancerous conditions often take the form of lysias containing dysplastic or hyperplastic cells. Accordingly, the presence of dysplasia or hyperplasia as an alternative or addition to the presence of cells with expressed markers or phenotypic characteristics of cancer stem cells can be used in the detection of precancerous conditions.

The severity of dysplasia can vary between different precancerous conditions, or with the development of one precancerous condition over time. Generally, the more advanced the dysplasia associated with a precancerous condition, the more likely it is that the precancerous condition will develop into cancer. Dysplasia is usually classified as mild, moderate, or severe. Severe diplasia usually progresses to cancer if left untreated. Accordingly, methods of therapeutic or medical uses using a compound of the present invention can therefore be used to treat a patient with a precancerous condition associated with severe dysplasia.

In a suitable embodiment of the present invention, the compound of the present invention is used to treat a patient with severe cervical dysplasia. Severe cervical dysplasia can be diagnosed with a cervical smear. In another embodiment of the present invention, a compound of the present invention is used to treat severe esophageal dysplasia (Barrett's syndrome). Severe esophageal dysplasia can be diagnosed after tissue biopsy.

It has recently been reported that precancerous conditions can also be identified by the detection of somatic mutations in cells in individuals who do not know they have cancer. In particular, age-related clonal hematopoiesis has been reported to be a common precancerous condition that is associated with increased overall mortality and increased risk of cardiometabolic disease. Most of the mutations found in blood cells occur in three genes: DNMT3A, TET2, and ASXL1. Accordingly, patients who will benefit from targeting cancer stem cells with a compound of the present invention and thereby treating a precancerous condition can be detected by analyzing a sample containing blood cells for the presence of precancerous genetic mutations of at least one of DNMT3A and / or TET2 and / or ASXL1.

Precancerous conditions for which treatment with a compound of the present invention according to the present invention to target cancer stem cells may be beneficial may also be detected by determining the presence of cancer stem cells with reference to any of the methods based on the expression of markers characteristic of cancer stem cells, or cancer stem cell phenotypes discussed elsewhere herein.

Cancer treatment

One skilled in the art will appreciate that there are many ways in which cancer treatment can be evaluated. Just by way of example, any mitigation or prevention of cancer development, cancer progression, cancer recurrence, or cancer spread can be considered indicative of effective cancer treatment.

In certain embodiments, a compound of the present invention can be used to reduce the proportion of cancer stem cells in a cancer cell population; and / or to inhibit tumor growth; and / or to reduce oncogenic potential; and / or to prevent or treat primary cancer; and / or to prevent or treat cancer recurrence; and / or for the prevention or treatment of metastatic or secondary cancer; and / or to treat, prevent or inhibit metastasis or recurrence; and / or to treat or prevent refractory cancer.

The ability to treat cancer by using a compound of the present invention resulting in a decrease in tumor size and also in maintaining a decrease in tumor size during / after the treatment period is a particularly significant indication of effective cancer treatment. As shown in the examples, the treatment or medical applications of the present invention are surprisingly effective in this aspect, even in models using cells representing a recurrent or refractory form of cancer that have previously been resistant to treatment with other therapies.

The data presented in the examples illustrate that treatment with a compound of the present invention reduces the proportion of cancer stem cells in the cancer cell population. Characteristic biological

- 11 036409 activities or cell surface markers that can detect cancer stem cells are described elsewhere in the present description. In a suitable embodiment, cancer treatment according to the present invention can result in a decrease in the proportion of cancer stem cells present in a patient with cancer by at least 10%, at least 20%, at least 30%, or at least by 40%. In suitable embodiments, cancer treatment according to the present invention can result in a decrease in the proportion of cancer stem cells present in a patient with cancer by at least 50%, at least 60%, at least 70%, or at least by 80%. Cancer treatment according to the present invention can result in a decrease in the proportion of cancer stem cells present in cancer in a patient by at least 85%, at least 90%, or at least 95%. Indeed, cancer treatment according to the present invention can result in a decrease in the proportion of cancer stem cells present in cancer in a patient by at least 96%, at least 97%, at least 98%, at least 99 % or even 100% (so there is essentially no cancer stem cell left).

Asymmetric division of cancer stem cells promotes tumor growth. Cancer treatment with a compound of the present invention according to the present invention can result in inhibition of tumor growth by at least 10%, at least 20%, at least 30%, or at least 40%. Accordingly, cancer treatment according to the present invention can result in inhibition of tumor growth by at least 50%, at least 60%, at least 70%, or at least 80%. Cancer treatment according to the present invention can result in inhibition of tumor growth by at least 85%, at least 90%, or at least 95% of the patient so treated. Indeed, cancer treatment according to the present invention can result in inhibition of tumor growth by at least 96%, at least 97%, at least 98%, at least 99% or even 100% of the treated cancer ...

Tumor growth can be assessed by any suitable method in which the change in tumor size is assessed over time. Accordingly, the size of a tumor before cancer treatment can be compared to the size of the same tumor during or after cancer treatment. A number of ways in which the size of a tumor can be estimated are known. For example, tumor size can be assessed by visualizing the tumor in situ in a patient. Suitable methods, such as imaging methods, can allow the determination of tumor volume and the assessment of changes in tumor volume.

As shown in the results provided in the examples of the present description, the methods of therapeutic and medical uses of the compounds of the present invention are capable of not only arresting tumor growth, but are indeed capable of reducing tumor volume in cancer patients, including patients with relapsed or refractory cancers. Accordingly, cancer treatment according to the present invention can result in a decrease in tumor volume by at least 10%, at least 20%, at least 30%, or at least 40%. In suitable embodiments, cancer treatment according to the present invention can result in a decrease in tumor volume by at least 50%, at least 60%, at least 70%, or at least 80%. Cancer treatment according to the present invention can result in a reduction in tumor volume of at least 85%, at least 90%, or at least 95%. Indeed, cancer treatment according to the present invention can result in a reduction in tumor volume of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

The reduction in tumor volume of the type described above can be calculated with reference to a suitable control. For example, in studies performed in vitro or in vivo in suitable animal models, the reduction in tumor volume can be determined by direct comparison between the volume of the tumor treated with a compound of the present invention and the volume of the control tumor (which may be untreated or that has been treated other than treatment with a compound of the present invention). It is clear that these models requiring no tumor treatment may be ethically unacceptable in the context of clinical trials or therapeutic treatment of patients, in which case the reduction in tumor volume can be assessed by comparing the volume of the tumor being treated with the same tumor before treatment or with the predicted volume. which the tumor could reach without treatment.

Methods for therapeutic and medical uses of a compound of the present invention can lead to a decrease in biomarkers indicative of cancer. Reducing these markers provides additional assessment by which an effective cancer treatment can be demonstrated. Suitable examples of these biomarkers can be selected based on the type of cancer to be treated for gynecological cancers, CA125 is a suitable example of a biomarker, while in the case of pancreatic cancer or liver cancer, CA19.9 is a suitable example of a biomarker, and in the case of colorectal cancer CEA can be a suitable biomarker.

Accordingly, cancer treatment according to the present invention can result in

- 12,036409 reduce cancer biomarkers by at least 10%, at least 20%, at least 30%, or at least 40%. In suitable embodiments, cancer treatment according to the present invention can result in a reduction of cancer biomarkers by at least 50%, at least 60%, at least 70%, or at least 80%. Cancer treatment according to the present invention can result in a reduction in cancer biomarkers of at least 85%, at least 90%, or at least 95%. Indeed, cancer treatment according to the present invention can result in a reduction in cancer biomarkers of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

The beneficial effects, such as a decrease in the proportion of cancer stem cells present, a slowdown in tumor growth, or a decrease in tumor volume or cancer biomarkers, observed in the cancer treatment of the present invention, can be sustained for at least one month. Accordingly, these beneficial effects can be sustained for at least two months, at least three months, at least four months, at least five months, or at least six months. Indeed, these beneficial effects can be sustained for at least 12 months, at least 18 months, or at least 24 months. Accordingly, the beneficial effects can be sustained for at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or ten years or more.

In a suitable embodiment of the present invention, a compound of the present invention is used in a method for the prevention or treatment of cancer or a precancerous condition by targeting cancer stem cells. In a suitable embodiment, the present invention relates to the use of a compound of the present invention in a method for preventing or treating cancer or a precancerous condition, wherein the method reduces the oncogenic potential of one or more cancer stem cells. Accordingly, these methods can prevent the development of cancer or inhibit tumor growth.

When a compound of the present invention is used in the methods or medical uses of the present invention to prevent or treat the development of cancer, the prevention or treatment can slow, delay, or completely arrest the progression of cancer.

Cancer progression is usually defined by assigning a stage to the cancer. Staging is usually accomplished by assigning the cancer a number I-IV, with I being isolated cancer and IV being cancer that has spread to the limit as measured by the score. The specificity of staging varies between types of cancer, but the stage usually takes into account the size of the tumor, whether it has invaded neighboring organs, how many regional (nearby) lymph nodes it has spread (if it has spread at all), and whether it is found in more distant places (metastasized) ...

Usually stage I is localized to one part of the body and can be treated with surgical removal (for solid tumors that are small enough). Stage II is locally advanced and is treated with chemotherapy, radiotherapy, surgery, or a combination of both. Stage III is also locally advanced, and the designation of stage II or stage III depends on the specific type of cancer, although stage III is generally accepted as late locally advanced. Stage IV cancer often metastasizes to a second organ. Cancer treatment using a compound of the present invention in the methods or medical uses of the present invention can be used to treat stage I, II, III, or IV cancers by targeting cancer stem cells. Treatment with a compound of the present invention can be used to prevent cancer progression from the first stage to the next. In one embodiment, treatment with a compound of the present invention is used to prevent progression from stage I to stage II. In another embodiment, treatment with a compound of the present invention is used to prevent progression from stage II to stage III. In yet another embodiment, treatment with a compound of the present invention is used to prevent progression from stage III to stage IV.

Preventing or inhibiting the progression of cancer is usually important in preventing the spread of cancer, for example, progression from stage I to stage II, where cancer spreads locally, or progression from stage III to stage IV, where cancer metastasizes to other organs. Cancer stem cells are tumor-forming and are therefore thought to play an important role in the spread of cancer, both locally and metastatically. The methods of therapeutic or medical uses of the present invention using a compound of the present invention can therefore be used to prevent the spread of cancer by targeting tumor-forming cancer stem cells and thus reducing their number.

Crayfish

Certain compounds of the present invention exhibit enhanced anti-cancer activity compared to the cladribine from which they are derived. This increase in anticancer activity appears to be due to increased activity against both cancer stem cells and non-stem cancer cells.

Cancer stem cells play a role in the biological activity of a wide range of cancer types. Accordingly, there is a wide range of cancer types that can be prevented or treated according to the present invention.

As discussed elsewhere herein, cancer stem cells are known to be present in many types of tumors, including liquid tissue tumors (including hematologic tumors such as leukemias and lymphomas) and solid tumors (such as breast, lung, colon , prostate, ovaries, skin, bladder, bile ducts and pancreas). Therefore, it is believed that methods of therapeutic and medical uses for a compound of the present invention to target cancer stem cells will be useful in preventing or treating these types of cancer.

Accordingly, the compound of the present invention can be used in the prevention or treatment of cancer selected from the group consisting of leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), squamous cell carcinoma of the mouth, bladder cancer, Leydig cell tumor, bile duct cancer such as cholangiocarcinoma or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer, and gynecological cancers including ovarian cancer, endometrial cancer, cancer fallopian tube, uterine cancer, and cervical cancer, including epithelial cancer of the cervix. In suitable embodiments, the cancer is leukemia, and may be selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia (also known as acute myelocytic leukemia or acute non-lymphocetic leukemia), acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia (also known as chronic myeloid leukemia, chronic myelocytic leukemia or chronic granulocytic leukemia), chronic lymphocytic leukemia, monoblastic leukemia, and hairy cell leukemia. In further preferred embodiments, the cancer is acute lymphoblastic leukemia. In a suitable embodiment, the cancer is a lymphoma that can be selected from the group consisting of Hodgkin's lymphoma; non-Hodgkin's lymphoma; Burkitt's lymphomas; and small cell lymphocytic lymphoma.

Accordingly, targeting cancer stem cells in these types of cancer can provide effective cancer treatment by preventing or treating cancer development, preventing or treating cancer progression, preventing or treating cancer recurrence, or preventing or treating cancer spread.

In a suitable embodiment, the present invention relates to a compound of the present invention for use in targeting cancer stem cells in the prevention or treatment of metastatic cancer.

In a suitable embodiment, the present invention relates to a compound of the present invention for use in targeting cancer stem cells in the treatment of recurrent or refractory cancer.

In a suitable embodiment, the present invention relates to a compound of the present invention for use in targeting cancer stem cells in the treatment of primary cancer. Accordingly, the primary cancer being treated may be the second primary cancer.

The present invention relates to a compound of the present invention for use in targeting cancer stem cells in the treatment of secondary cancer. In a suitable embodiment, the secondary cancer is metastatic cancer.

In a suitable embodiment, the present invention relates to a compound of the present invention for use in targeting cancer stem cells, wherein targeting cancer stem cells prevents or inhibits: (i) cancer recurrence; (ii) the occurrence of a second primary cancer; or (iii) cancer metastases.

Methods of therapeutic or medical applications in which a compound of the present invention is used based on its ability to target cancer stem cells can be used in the treatment of recurrent or refractory cancers. The considerations regarding recurrent or refractory cancer in these embodiments are, unless the context otherwise requires, the same as for the treatment of recurrent or refractory cancer in connection with other aspects of the present invention.

Recurrent or refractory cancer

As noted above, certain aspects and embodiments of the present invention relate, in particular, to the use of a compound of the present invention in the treatment of recurrent or refractory cancers.

For purposes of the present invention, refractory cancers can be mistaken for cancers that exhibit resistance to treatment with anticancer therapies other than therapies using a compound of the present invention. For example, a compound of the present invention can

- 14 036409 used in the treatment of refractory cancers that are resistant to radiotherapy treatment. Alternatively or additionally, the compound of the present invention can be used in the treatment of refractory cancers that are resistant to biological agents used in the treatment of cancer. In a suitable embodiment, the compound of the present invention can be used in the treatment of refractory cancers that are resistant to treatment with a chemotherapeutic agent other than the compound of the present invention.

In particular, refractory cancers that can benefit from the treatments and medical uses of the present invention using a compound of the present invention include cancers that are resistant to cladribine.

Recurrent cancers (or recurrent cancers) are cancers that reappear after a period of remission during which the cancer could not be detected. Cancer recurrence can occur at the site of the original cancer (local recurrence of cancer), at a site close to the site of the original cancer (regional recurrence of cancer), or at a place farther from the site of the original cancer (distant recurrence of cancer). Cancer stem cells are thought to play a role in cancer recurrence by providing a source from which recurrent cancer cells are generated. Accordingly, methods of therapeutic and medical uses of a compound of the present invention according to the present invention, which provide targeting of cancer stem cells, can be very useful in the context of recurrent cancer. The ability of a compound of the present invention to target cancer stem cells can be used to remove populations of these cells that are capable of causing relapse, thereby preventing recurrence of cancer. Anti-Cancer Stem Cell Activity The compounds of the present invention can also be used to target cancer stem cells in cancer that recurs, as well as potentially manifest cytotoxic effects on non-stem cancer cells, thereby providing treatment for recurring cancer.

In view of the above, it is clear that the compound of the present invention can be used in the methods or uses of the present invention to prevent or treat recurrent cancer. The compound of the present invention can be used in the methods or uses of the present invention to prevent or treat local, regional, or remote recurrent cancer.

The compound of the present invention can be used in the methods or uses of the present invention to prevent cancer recurrence by providing at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least at least 30 months of remission. Indeed, the compound of the present invention can be used to prevent cancer recurrence by providing at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least at least 10 years of remission.

The compound of the present invention can be used in the methods or uses of the present invention for the treatment of recurrent cancer that has recurred after at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months of remission. Indeed, the compound of the present invention can be used to treat recurrent cancer that has recurred after at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years or at least 10 years of remission.

The ability of the compounds of the present invention to target cancer stem cells confers the ability of the compounds to prevent or treat cancer in accordance with the medical uses or methods of treatment of the present invention. However, it should be noted that the compounds of the present invention also exhibit a direct cytotoxic effect on non-stem cancer cells that form tumor volume. While the activity of cancer stem cells may underlie much of the resistance that makes recurrent or refractory cancers so difficult to treat, non-stem cancer cells are also a major component of this recurrent or refractory cancers.

Certain compounds of the present invention exhibit greater cytostatic effects on non-stem cancer cells than does cladribine, the chemotherapeutic molecule from which the compounds of the present invention are derived.

Accordingly, the mechanism by which the compound of the present invention acts in the treatment of recurrent or refractory cancers may not be limited only to this activity against cancer stem cells of the compound, but can also apply the effect of the compound of the present invention on non-stem cancer cells. In these applications, treatment with a compound of the present invention will reduce the total number of both cancer stem cells and non-stem cancer cells, but will preferably reduce the proportion of cancer stem cells that remain after treatment.

Therapeutically Effective Doses of a Compound of the Present Invention

A therapeutically effective amount of a compound of the present invention may be

- 15 036409 is an amount sufficient to cause the death of cancer cells. A therapeutically effective amount of a compound of the present invention may be an amount sufficient to cause the death of cancer stem cells. In some embodiments, especially embodiments relating to the treatment of recurrent or refractory cancers, a therapeutically effective amount of a compound of the present invention may be an amount sufficient to cause the death of cancer stem cells and also to cause the death of non-stem cancer cells ...

There are various excellent methods in which the amount of a therapeutically effective compound, such as a compound of the present invention, that will be administered to a patient can be calculated and recalculated. One given method, which is considered particularly suitable for doses of agents for preventing or treating cancer, comprises the amount of agent to be administered per unit surface area of the patient's body. These doses are usually expressed in terms of the amount of agent (which can be determined by weight) per square meter (m ² ) surface area.

Throughout the description and claims of the present invention, the words includes and contains and their variations are meant including, but not limited to, and they are not intended (and really are not) excluding other fragments, additives, components, whole or steps. Throughout the description and claims of the present invention, the unitary forms include the plural, unless the context requires otherwise. In particular, when the indefinite article is used, the description should be interpreted to imply the plural form, as well as the singular form, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the present invention are to be interpreted as applicable to any other aspect, embodiment, or example described in the present invention unless they are incompatible. ... All of the features described in the present invention (including any appended claims, abstract and drawings), and / or all steps of any of the methods or processes thus described, can be combined in any combination, except for combinations where at least , some of these features and / or stages are mutually exclusive. The present invention is not limited to the details of any of the above embodiments. The present invention extends to any new feature or any new combination of features described in the present invention (including any appended claims, abstract and drawings), or to any new step or any new combination of steps in any method or process so described.

The attention of the reader is directed to all articles and documents that are filed simultaneously or prior to this description relating to this application, and which are in the public domain with this description, and the contents of all these articles and documents are incorporated into the present invention by reference.

Examples of

Example 1. Methods of obtaining.

Throughout the description, the following abbreviations have the meanings indicated:

DCM for dichloromethane;

DMF-N, N is dimethylformamide;

TBDMS, tert-butyldimethylsyl;

TFA, trifluoroacetic acid;

THF - tetrahydrofuran;

MeOH - methanol;

br s - broadened singlet;

tBuMgCl - tert-butyl magnesium chloride.

'-O-tert-Butyldimethylsilylcladribine A.

Cladribine (0.25 g, 0.88 mmol) was dissolved in anhydrous DMF (10 ml), TBDMSCl (0.15 g, 1.03 mmol) and imidazole (0.26 g, 3.82 mmol) were added under argon ... The mixture was stirred at room temperature for 24 hours before adding water. The precipitate was filtered off and purified by silica gel column chromatography to give 3 ', 5'-bis-O-tert-butyldimethylsilylcladribine (0.12 g, 27%) and the desired product (0.12 g, 34%).

¹ H NMR (500 MHz, DMSO): δ 8.28 (1H, s, H-8), 7.78 (2H, br s, NH ₂ ), 6.26 (1H, t, J = 6.6 Hz, H-1 '), 5.39 (1H, br s, 3'-OH), 4.40 (1H, m, H-3'), 3.87 (1H, m, H-4 ') , 3.84-3.68 (2H, m, 2 x H-5 '), 2.69, 2.33 (2H, 2 x m, 2 x H-2') 0.85 (9H, s, C (CH3) h), 0.02 (6H, s, Si (CH ₃ ) ₂ ).

Standard method A.

Protected nucleoside A (78 mg, 0.20 mmol) was dissolved in anhydrous tetrahydrofuran under argon and 1M tBuMgCl in THF (0.4 ml, 0.4 mmol) was added dropwise, followed by the slow addition of the desired phosphorochloridate (0.4 mmol ) in anhydrous THF. The mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by column chromatography on silica gel with 3% MeOH in DCM as eluent. The resulting compound was dissolved in H ₂ O / THF (1: 1) and TFA was added dropwise. The mixture was stirred at room temperature for

- 16 036409 min and added to neutralize the NaHCO ₃ solution. The solvents were evaporated and the residue was purified by silica gel column chromatography and preparative thin layer chromatography (TLC) (10%

MeOH in DCM).

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (ethoxy-L-alanyl)] phosphate 1.

H? N n

N 1 \.? i Cl

HO ... _N N o o

II ⁿ

Et A Ν P — O

Prepared according to standard method A using 5'-protected cladribine A (0.1 g, 0.17 mmol) in anhydrous THF, tBuMgCl (1M THF solution, 0.34 ml, 0.34 mmol), phenyl (ethoxy-balanyl ) phosphorochloridate (0.10 g, 0.34 mmol). After deblocking with TFA, the crude mixture was purified by column chromatography (4% MeOH in DCM) followed by preparative TLC (4% MeOH in DCM) to give the pure product as a white solid (19.0 mg, 21%). ³¹ P-NMR (202 MHz, CD3OD): δ 3.28, 2.76. ¹ H-NMR (500 MHz, CD3OD): δ 8.32, 8.28 (1H, 2 x s, H-8), 7.41-7.22 (5H, m, H-Ar), 6, 43, 6.33 (1H, 2 x t, J = 7.0 Hz, H-1 '), 5.3, 5.29 (1H, 2 x br s, H-3'), 4.32, 4.31 (1H, 2 x br s, H-4 '), 4.22-4.15 (2H, m, CH2CH3), 4.05-4.00 (1H, m, NHCHCH3), 3.86 -3.80 (2H, m, 2 x H-5 '), 3.01-2.90 (1H, m, H-2'а), 2.812.78 (0.5H, m, H-2' _b one diastereoisomer), 2.68-2.64 (0.5H, m, H-2 ' _b one diastereoisomer), 1.42-1.36 (3H, 2 x d, J = 7.0 Hz, NHCHCH3 ), 1.30-1.24 (3H, m, CH ₂ CH ₃ ). MS (ES +) m / z: 563.1 (M + Na +, 100%); exact mass: C ₂₁ H ₂₆ ClN ₆ O ₇ NaP desired m / z 563.1187, found m / z 563.1183. Reverse phase HPLC eluting with H ₂ O / CH ₃ CN from 100/0 to 0/100 for 20 min, flow = 1 ml / min, λ = 254, t _R = 12.31, 12.41 min.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (tert-butoxy-L-alanyl)] phosphate 2.

Prepared according to standard method A using 5'-protected cladribine A (0.10 g, 0.17 mmol) in dry THF, tBuMgCl (1M THF solution, 0.34 ml, 0.34 mmol), phenyl (tert-butoxy Β-alanyl) phosphorochloridate (0.11 g, 0.34 mmol). After deblocking with TFA, the crude residue was purified by column chromatography (4% MeOH in DCM) to give the pure product as a white solid (43.8 mg, 45%). 31P-NMR (202 MHz, CD3OD): δ 3.30, 2.91; 1H-NMR (500 MHz, CD3OD): δ 8.31, 8.27 (1H, 2 x s, H-8), 7.42-7.21 (5H, m, H-Ar), 6.43 , 6.34 (1H, 2 x t, J = 7.0 Hz, H-1 '), 5.36, 5.30 (1H, 2 x br s, H-3'), 4.35, 4 , 30 (1H, 2 x br s, H-4 '), 3.91-3.80 (3H, m, NHCHCH3, 2 x H-5'), 3.02-2.90 (1H, m, H-2 'a), 2.81-2.77 (0.5H, m, H-2' _b one diastereoisomer), 2.68-2.65 (0.5H, m, H-2 ' _b one diastereoisomer), 1.49, 1.46 (9H, 2 x s, C (CH3) h), 1.38, 1.34 (3H, 2 x d, J = 7.0 Hz, NHCHCH3). MS (ES +) m / z: 591.1 (M + Na +, 100%); exact mass: C ₂ 3H ₃₀ CIN ₆ O ₇ aP required m / z 591.1500, m / z found 591.1509. Reverse phase HPLC eluting with H2O / CH3CN from 100/0 to 0/100 for 20 min, flow = 1 ml / min, λ = 254, t _R = 15.24, 15.36 min.

- 17 036409

2-Chloro-2'-deoxyadenosine-3'- [phenyl (oenzoxy-O-alanyl)] phosphate 3.

H ₂ N

Prepared according to standard method A using 5'-protected cladribine A (0.11 g, 0.19 mmol) in anhydrous THF, tBuMgCl (1M THF solution, 0.37 ml, 0.37 mmol), phenyl (benzoxy-Ealanyl ) phosphorochloridate (0.13 g, 0.37 mmol). After deblocking with TFA, the crude residue was purified by column chromatography (4% MeOH in DCM) to give the pure product as a white solid (40.0 mg, 35%). ³¹ P-NMR (202 MHz, CD ₃ OD): δ 3.06, 2.77. 1H-NMR (500 MHz, CD3OD): δ 8.24, 8.22 (1H, 2 x s, H-8), 7.39-7.16 (10H, m, H-Ar), 6.32 -6.26 (1H, m, H-1 '), 5.34-5.32, 5.23-5.21 (1H, 2 x m, H-3'), 5.195.14 (2H, m , CH ₂ Ph), 4.36-4.34 (0.5H, m, H-4 'one diastereoisomer), 4.22-4.20 (0.5H, m, H-4' one diastereoisomer) 4 , 10-4.04 (1H, m, NHCHCH3), 3.85-3.72 (2H, m, 2 x H-5 '), 2.87-2.82, 2.77-2.67 ( 1H, 2 x m, H2'а), 2.59, 2.48 (1H, 2 x ddd, J = 14.0, 5.8, 1.9 Hz, H-2'b), 1.41 , 1.38 (3H, 2 x dd, J = 7.0, 4.5 Hz, NHCHCH3). MS (ES +) m / z: 625.1 (M + Na +, 100%); exact mass: C ₂ 6H ₂₈ CIN ₆ O ₇ NaP, m / z required 625.1343, found m / z 625.1351. Reverse phase HPLC eluting with H2O / CH3CN from 100/0 to 0/100 for 20 min, flow = 1 ml / min, λ = 254, t _R = 14.16 min.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxyglycinyl)] phosphate 4.

Prepared according to standard method A using 5'-protected cladribine A (0.12 g, 0.20 mmol) in anhydrous THF, tBuMgCl (1M THF solution, 0.40 ml, 0.40 mmol), phenyl (benzoxy-L glycinyl ) phosphorochloridate (0.14 g, 0.40 mmol). After deblocking with TFA, the crude residue was purified by column chromatography (4% MeOH in DCM) to give the pure product as a white foamy residue (15.0 mg, 13%). ³¹ P-NMR (202 MHz, CD3OD): δ 4.21, 4.05. 'H-NMR (500 MHz, CD3OD): δ 8.14, 8.13 (1H, 2 x s, H-8), 7.29-7.08 (10H, m, H-Ar), 6, 23-6.16 (1H, m, H-1 '), 5.24-5.21 (1H, m, H-3'), 5.085.06 (2H, m, CH ₂ Ph), 4.20 -4.19 (0.5H, m, H-4 'one diastereoisomer), 4.14-4.12 (0.5H, m, H-4' one diastereoisomer), 3.77-3.63 (4H , m, NHCH2, 2 x H-5 '), 2.76-2.70 (1H, m, H-2'), 2.51-2.47 (1H, m, H-2 '). MS (ES +) m / z: 611.1 (M + Na +, 100%); Exact mass: C ₂₅ H ₂₆ CIN ₆ O ₇ NaP, m / z required 611.1187, found m / z 611.1180. Reverse phase HPLC eluting with H2O / CH3CN from 100/0 to 0/100 for 20 min, flow = 1 ml / min, λ = 254, t _R = 13.64 min.

Additional compounds of the present invention can be prepared according to standard method A, analogously to compounds 1-4. Examples include the following compounds.

- 18 036409

2-Chloro-2'-deoxyadenosine-3'- [phenyl (benzoxy-L-leucinyl)] phosphate 5.

³¹ P-NMR (CD3OD, 202 MHz): δ 3.58, 2.81; 1H-NMR (CD ₃ OD, 500 MHz): δ 8.28, 8.23 (1H, 2 x s, H-8), 7.40-7.22 (10H, m, H-Ar), 6 , 36, 6.25 (1H, 2 x t, J = 7.0 Hz, H-1 '), 5.30, 5.24 (1H, 2 x br s, H-3'), 5.19 -5.14 (2H, m, CHPh), 4.25 (1H, br s, H-4 '), 3.98-3.95 (1H, m, NHCH), 3.84-3.73 ( 2H, m, 2 x H-5 '), 2.89-2.55 (2H, m, 2 x H-2'), 1.77-1.72 (1H, m, CH (CH3E), 1 , 60-1.56 (2H, m, C ^ CHCHCH3E), 0.94-0.88 (6H, m, CH (CH3E). MS (ES +) m / z: 667.2 (M + Na +, 100 %); exact mass: C ₂ 9H ₃₄ CIN ₆ O ₇ NaP, required m / z 667.1813, found m / z 667.1799 Reverse phase HPLC, eluting H2O / CH3CN 100/0 to 0/100 in for 20 min, flow = 1 ml / min, λ = 254, t _R = 16.27, 16.47 min.

2-Chloro-2'-deoxyadenosine-3 '- [1-naphthyl- (2,2-dimethylpropoxy-L-alanyl)] phosphate 6.

³¹ P-NMR (202 MHz, CD3OD): δ 3.68, 3.27. 1H-NMR (500 MHz, CD3OD): δ 8.28-8.20 (2H, m, H-8, H-Ar), 7.92 (1H, d, J = 8.0 Hz, H-Ar ), 7.75 (1H, d, J = 8.2 Hz, H-Ar), 7.63-7.47 (4H, m, H-Ar), 6.38-6.21 (1H, 2 x m, H-1 '), 5.39-5.33 (1H, 2 x m, H-3'), 4.32-4.23 (1H, 2 x m, H-4 '), 4 , 17-4.11 (1H, m, NHCHCH3), 3.89-3.71 (4H, m, 2 x H-5 ', CH ₂ C (CH _h ) _h ), 2.96-2.78 , 2.58-2.55 (2H, 2 x m, 2 x H-2 '), 1.43, 1.38 (3H, 2 x d, J = 7.25 Hz, NHCHCH3), 0.95 , 0.93 (2 x s, 9H, CH ₂ C (CH _h ) _h ). MS (ES +) m / z: 656 (M + Na +), 634 (M + H +); C ₂₈ H ₃₄ CIN ₆ O ₇ P Required weight 633.03.

2-Chloro-2'-deoxyadenosine-3 '- [1-naphthyl (pentoxyA-leucinyl)] phosphate 7.

³¹ P-NMR (202 MHz, CD3OD): δ 4.02, 3.48. 1H-NMR (500 MHz, CD3OD): δ 8.24, 8.21 (1H, 2 x s, H-8), 7.94, 7.92 (1H, 2 x s, H-Ar), 7 , 76, 7.53 (1H, 2 x s, H-Ar), 7.63-7.55 (4H, m, H-Ar), 7.52, 7.47 (1H, 2 x s, H -Ar), 6.40-6.33, 6.23-6.20 (1H, 2 x m, H-1 '), 5.40-5.38, 5.32-5.29 (1H, 2 x m, H-3 '), 4.34-4.33, 4.26-4.24 (1H, 2 x m,

- 19 036409

H-4 '), 4.08-4.05 (2H, m, 2 x H-5'), 4.01-3.97 (1H, m, NHCH ^ C ^^ k), 3.88- 3.80 (2H, m,

NECH ^ C ^ C ^^), 3.00-2.95, 2.89-2.79, 2.58-2.54 (2H, 3 x m, 2 x H-2 '), 1.75 -1.69 (1H, m, NHCHCH ^ H ^^ h), 1.63-1.55 (4H, m, 2 x CH ₂ , O CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 1.35 -1.28 (4H, m, 2 x CH ₂ , OCH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 0.91-0.82 (9H, m, OCH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , NHCHCH ₂ CH (CH ₃ ) ₂ ). MS (ES +) m / z: 697 (M + Na +), 675 (M + H +), C ₃ iH ₄₀ CIN ₆ O ₇ P required mass 674.24.

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl (cyclohexoxy-L-alanyl)] phosphate 8.

³¹ P-NMR (202 MHz, CD3OD) δ 3.34, 2.80. -NMR (500 MHz, CD3OD): δ 8.33-8.28 (2H, m, H-8, H-Ar) 7.85-7.81 (1H, m, H-Ar, 7.62 -7.59 (1H, m, H-Ar), 7.56-7.52 (3H, m, H-Ar), 7.47-7.43 (1H, m, H-Ar), 6, 30 (1H, t, J = 6.5 Hz, H-1 '), 4.66-4.57 (3H, m, H-3', OCH-ether), 4.51-4.48 (2H , m, 2 x H-5 '), 4.20-4.18 (1H, m, H-4'), 4.123.96 (1H, m, NHCHCH ₃ ), 2.67-2.54 (1H , m, H-2 '), 2.50-2.46 (1H, m, H-2'), 1.75-1.71 (4H, m, 2 x Cd-ether), 1.32 -1.30 (9H, m, 3x (CHE-ether. NHCHCH3). MS (ES +) m / z: 667 (M + Na +), 645 (M + H +); C ₂₉ H ₃₄ CIN ₆ O ₇ P, required mass 644.19.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (cyclohexoxy-L-alanyl)] phosphate 9.

³¹ P-NMR (202 MHz, CD3OD): δ 3.34, 2.80. ¹ H-NMR (500 MHz, CD3OD): δ 8.32-8.28 (1H, m, H-8), 7.437.37 (2H, m, H-Ar), 7.31-7.22 ( 3H, m, H-Ar), 6.44-6.41, 6.36-6.29 (1H, 2 x m, H-1 '), 5.37-5.34, 5.30-5 , 27 (1H, 2 x m, H-3 '), 4.81-4.74 (1H, m, CH-ether), 4.33-4.25 (1H, m, H-4'), 4.01-3.92 (1H, m, NHCHCH3), 3.87-3.79 (2H, m, 2 x H-5 '), 3.01-2.89 (1H, m, H-2 '), 2.82-2.77, 2.67-2.63 (1H, 2 x m, H-2'), 1.86-1.74 (4H, m, 2 x OD ether), 1.57-1.30 (9H, m, 3 x Cd-ether, NHCHCH3). MS (ES +) m / z: 617 (M + Na +), 595 (M + H +); C25H32CIN6O7P, required mass 594.18.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (2,2-dimethylpropoxy-E-alanyl)] phosphate 10.

- 20 036409 ³¹ P-NMR (202 MHz, CD3OD): δ 3.30, 2.76. Γ-NMR (500 MHz, H CD ₃ OD): δ 8.32, 8.27, 8.23, 8.07 (1H, 4 x s, H-8), 7.57-7.52, 7 , 43-7.38, 7.31-7.22 (5H, 3 x m, H-Ar), 6.44-6.41, 6.36-6.30 (1H, 2 x m, H- 1 '), 5.38-5.36, 5.30-5.27 (1H, 2 x m, H-3'), 4.32-4.30, 4.27-4.25 (1H, 2 x m, H-4 '), 4.15-4.12, 4.08-4.04 (1H, 2 x m, NHCHCH3), 3.87-3.79 (1H, m, H-5 '), 3.92-3.80 (2H, m, SSCHSCH3b), 3.68, 3.58 (1H, 2 x dd, J = 12.0, 5.0 Hz, H-5'), 3 , 01-2.89, 2.81-2.64, 2.35-2.29 (2H, 3 x m, 2 x H-2 '), 1.44, 1.39 (3H, 2 x d , J = 7.5 Hz, NHCHCH3), 0.98, 0.96 (9H, 2 x s, CH ₂ C (CH _h ) _h ). MS (ES +) m / z: 605 (M + Na +), 583 (M + H +), C ₂₄ H ₃₂ CIN ₆ O ₇ P, required mass 582.18.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (ethoxy-2,2-dimethylglycinyl)] phosphate 11.

³¹ P-NMR (202 MHz, CD3OD): δ 1.60, 1.55. 1H-NMR (500 MHz, CD3OD): δ 8.32, 8.28 (1H, 2 x s, H-8), 7.45-7.38, 7.33-7.27, 7.24- 7.21 (5H, 3 x m, H-Ar), 6.41, 6.33 (1H, 2 x dd, J = 8.1, 5.8 Hz, H-1 '), 5.36- 5.32 (1H, m, H-3 '), 4.39, 4.29 (1H, 2 x m, H-4'), 4.20, 4.19 (2H, 2 x q, J = 7.2 Hz, CH2CH3), 3.92-3.76 (2H, m, 2 x H-5 '), 3.002.88, 2.78-2.63 (2H, 3 x m, 2 x H- 2 '), 1.53 (6H, br s, (CH3) ₂ ), 1.29, 1.28 (3H, 2 x t, J = 7.1 Hz, CH2CH3). MS (ES +) m / z: 577.7 (M + Na +), C22H28CIN6O7P, required mass 554.92.

2-Chloro-2'-deoxyadenosine-3'- [phenyl (oenzoxy-E-phenylalanyl)! phosphate 12.

³¹ P-NMR (202 MHz, CDCl3): δ 1.42, 1.23. 1H-NMR (500 MHz, CDCI3): δ 7.69, 7.55 (1H, 2 x s, H-8), 7.286.92 (15H, m, H-Ar), 6.11 (2H, br s, NH2), 6.01-5.86 (1H, m, H-1 '), 5.30-5.02 (4H, m, H-3', OH-3 ', OCH2Ph), 4, 31-4.15 (2H, m, H-4 ', NHCHCHPh, 3.86-3.65 (3H, m, 2 x H-5', NHCHCHPh), 2.98-2.81 (3H, m , NHCHCHPh, H-2a '), 2.39-2.31 (1H, m, H-2b'). MS (ES +) m / z: 679 [M + H +], 681 [M ( ³⁷ C1) + H +], 701 [M + Na +], 703 [M ( ³⁷ C1) + Na +], 717 [M + K +], 719 [M ( ³⁷ C1) + K +]; C32H32CIN6O7P, required mass m / z 678.18 Reverse phase HPLC eluting with H2O / CH3CN 100/0 to 0/100 for 10 min, flow = 1 ml / min, λ = 254, tR = 9.19 min.

- 21 036409

2-Chloro-2'-deoxyadenosine-3'- [1-naphthyl (benzoxy-E-phenylalanyl)] phosphate 13.

³¹ P-NMR (202 MHz, CDCl3): δ 2.09, 1.78. 1H-NMR (500 MHz, CDCl3): δ 8.02-7.91 (1H, m, H-8), 7.817.79 (1H, m, H-Ar), 7.65-7.60 (1H , m, H-Ar), 7.49-7.41 (4H, m, H-Ar), 7.33-6.75 (11H, m, H-3 Naph, OCH + li. CHCHPh), 6 , 00 (2H, br s, NH), 5.92-5.62 (1H, 2 m, H-1 '), 5.28-5.13 (2H, m, H-3' OH-3 ' ), 5.05-4.89 (2H, m, OCHPh), 4.34-4.27 (1H, m, NHCHCH2Ph), 4.18-4.12 (1H, 2 m, H-4 ') 3.83-3.61 (3H, m, 2 x H-5 ', NHCHCHPh), 2.95-2.88 (2H, m, NHCHCHPh), 2.84-2.79 (1H, m, H -2'a), 2.30-2.14 (1H, m, H-2'b). MS (ES ⁺ ) m / z: 729 | M + H '|, 751 | M + Na' |, 767 [M + K ⁺ ]; Exact mass: C36H ₃₄ CIN ₆ O ₇ P required m / z 728.19. Reverse phase HPLC H2O / CH3CN from 100/0 to 0/100 for 10 min, flow = 1 ml / min, λ = 254, t _R = 10.33 min.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxy-E-valinyl)] phosphate 14.

³¹ P-NMR (202 MHz, CDCl ₃ ): δ 2.40, 2.20. 'H-NMR (500 MHz, CDCl ₃ ): δ 7.74, 7.58 (1H, 2 x s, H-8), 7.30

7.09 (10H, m, H-Ar), 6.13-6.11, 6.27-6.24 (1H, 2 x m, H-1 '), 5.91 (2H, br s, NH), 5.27-5.25 (1H, m, H-3 '), 5.105.04 (2H, m, CHPh), 4.25-4.21 (1H, m, H-4'), 3.86-3.62 (4H, m, 2 x H-5 ', NHCHCH (CH3) 2, NHCHCH (CH ₃ ) 2,), 2.95-2.90 (1H, m, H-2' a), 2.45-2.37 (1H, m, H-2'b), 2.02-1.99 (1H, m, NHCHCH (CH ₃ ) ₂ ), 0.88-0.77 ( 6H, m, NHCHCH (CH ₃ ) ₂ ). MS (ES +) m / z: 632 [M + H ⁺ ], 655 [M + Na ⁺ ]. C ₂₈ H ₃₂ CIN ₆ O ₇ P required mass m / z 631.18.

2-Chloro-2'-deoxyadenosine-3'- [phenyl (isopropoxy-E-alanyl)] phosphate 15.

³¹ P-NMR (202 MHz, CDCl ₃ ): δ 1.54, 1.34. S-NMR (500 MHz, CDCl ₃ ): δ 7.79, 7.71 (1H, 2s, H-8), 7.317.12 (5H, m, H-Ar), 6.21-6.17, 6.05-6.01 (1H, 2 x m, H-1 '), 5.70 (2H, br s, NH), 5.31-5.26 (1H, m, H-3'), 4.994.93 (1H, m, OCH (CH ₃ ) 2), 4.44-4.42 (1H, m, H-4 '), 3.98-3.87 (2H, m, NHCHCH3, OH- 5 '), 3.82-3.74 (1H, m,

- 22 036409

NHCHCH3), 3.67-3.53 (3H, m, 2 x H-5 ', NHCHCH3), 3.08-2.97 (1H, m, H-2'), 2.62 -2.49 (1H, m, H-2 '), 1.34,

1.30 (3H, 2 x d, J = 7.0 Hz, NHCHCH3), 1.22-1.19 (6H, m, OCH (CH3E). MS (ES +) m / z: 555 [M + H +], 556

[M ( ¹³ C) + H +], 557 [M ( ³⁷ a) + H], 558 [M ( ³⁷ O, ¹³ C) + H +], 577 [M + Na +], 578 [M ( ¹³ G ) + Na +], 579 [M ( ³⁷ Cl) + Na +],

580 [M ( ³⁷ Cl, ¹³ Q + Na ⁺ ]. C ₂₂ H ₂₈ CIN ₆ O ₇ P, desired mass m / z 554.14. Reversed phase HPLC, eluting

H2O / CH ₃ CN from 100/0 to 0/100 for 15 min, flow = 1 ml / min, λ = 254, t _R = 9.92 min.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (2-butoxy-L-alanyl)] phosphate 16.

31P-NMR (202 MHz, CDC13): δ 1.62, 1.60. 1 H-NMR (500 MHz, CDCl ₃ ): δ 7.81, 7.73 (1H, 2 x s, H-8), 7.307.11 (5H, m, H-Ar), 6.21-6, 03 (3H, m, H-1 ', NH), 5.32-5.23 (2H, m, H-3', NHCHCH3), 4.85-4.76 (1H, m, OCH (CHs) CH ₂ CH ₃ ), 4.34-4.31 (1H, m, H-4 '), 3.95-3.75 (4H, m, NHCHCH3, 2 x H-5', OH-5 ') , 3.07-2.95 (1H, m, H-2 _a '), 2.48-2.26 (1H, m, H-2b'), 1.55-1.47 (2H, m, OCH (CH ₃ ) CH2CH3), 1.37-1.31 (3H, m, NHCHCH3), 1.191.09 (3H, m, OCH (CH ₃ ) CH2CH3), 0.85-0.79 (3H, m , OCH (CH ₃ ) CH2CH3). MS (ES +) m / z: 569 [M + H +], 570 [M ( ¹³ C) + H +], 571 [M ( ³⁷ C1) + H +], 572 [M ( ³⁷ a, ¹³ C) + H +], 591 [M + Na +], 592 [M ( ¹³ C) + Na +], 593 | М (3 Cl) iNa '|, 594 [М ( ³⁷ С1, ¹³ O + Na +], 607 [М + К +], 608 [М ( ¹³ С) + К +], 609 [М ( ³⁷ С1) + K +]. C ₂₃ H ₃₀ CIN ₆ O ₇ P required mass m / z 568. Reversed phase HPLC, eluting with H2O / CH3CN from 100/0 to 0/100 for 15 min, flow = 1 ml / min, λ = 254, t _R = 10.48 min.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (^) - 1-phenylethoxy-E-alanyl) phosphate 17.

31P-NMR (202 MHz, CDC13): δ 3.34, 2.65. 1H-NMR (500 MHz, CDC13): δ 8.20 (1H, m, H-8), 7.47-7.28 (8H, m, H-Ar), 7.27-7.15 (2H , m, H- Ar), 6.40-6.33, 6.32-6.26, (1H, 2 x m, H-1 '), 5.96-5.84 (1H, m, CHCH3Ph ), 5.35-5.21 (1H, m, H-3 '), 4.40-4.26 (1H, m, H-4'), 4.10-4.02 (1H, m, NHCHCH3), 3.87-3.74 (2H, m, 2 x H-5 '), 2.94-2.83 (1H, m, H-2a'), 2.74-2.57 (1H , m, H-2 _b '), 1.60-1.48 (3H, m, CHCH3RK), 1.43-1.20 (3H, m, NHCHCH3). MS (ES +) m / z: 639 [M + Na +], 640 [M ( ³⁷ C1) + # +]. C ₂₇ H ₃₀ CIN ₆ O ₇ PNa required mass m / z 639.99. Reverse phase HPLC, eluting with H ₂ O / MeOH from 100/0 to 0/100 for 35 min, flow = 1 ml / min, λ = 254, tR = 29.19, 29.54 min.

- 23 036409

2-Chloro-2'-deoxyadenosine-3 '- [1-naphthyl (benzoxy-E-alanyl) phosphate 18.h ₂ n

Protected nucleoside A (78 mg, 0.20 mmol) was dissolved in anhydrous THF (7 ml) under argon and tBuMgCl (1.0 M in THF, 1.0 ml, 1.0 mmol) was added dropwise, followed by slow adding 1-naphthyl- (benzoxy-E-alanyl) -phosphorochloridate (0.40 g, 0.99 mmol) in anhydrous THF (2.0 ml). The mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by silica gel column chromatography (3% MeOH in DCM). The resulting compound was dissolved in H ₂ O / THF (4 ml + 4 ml) and TFA (1 ml) was added dropwise. The mixture was stirred at room temperature for 30 min and NaHCO _{3 was} added to neutralize the solution. The solvents were evaporated and the residue was purified by silica gel column chromatography (0-4% MeOH in DCM) and preparative TLC (10% MeOH in DCM). Yield: 50 mg. 38% at 2 stages. 31P-NMR (202 MHz, CD3OD): δ 3.71, 3.10. S-NMR (500 MHz, CD ₃ OD): δ 8.17, 8.14 (2H, 2 x s, H-8, H-Ar), 7.88 (1H, d, J = 7.7 Hz , H- Ar), 7.71 (1H, dd, J = 8.1, 4.2 Hz, H- Ar), 7.59-7.41 (4H, m, H-Ar), 7.35 -7.16 (5H, m, H-Ar), 6.30, 6.15 (1H, 2 x dd, J = 8.4, 5.9 Hz, H-1 '), 5.33 (1H , m, H-3 '), 5.18-5.06 (2H, m, CH2Ph), 4.27-4.24, 4.21-4.12 (2H, 2 x m, H-4' , NHCHCH3), 3.84-3.67 (2H, m, 2 x H-5 '), 2.82-2.78 (1H, m, C-2D 2.69-2.65, 2.64 -2.61 (1H, 2 x m, H-2b '), 1.40, 1.35 (3H, 2 x d, J = 7.2 Hz, NHCHCH3). MS (ES +) m / z: 676 , 16 (M + Na +). C ₃₀ H ₃₀ CIN ₆ O ₇ Pna, required mass m / z 676.02 Reversed phase HPLC, eluting with H2O / MeOH from 100/0 to 0/100 for 40 min, flow = 1 ml / min, λ = 254, t _R = 20.6, 21.1 min.

2-Chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxy-E-alanyl) phosphate 19.

Protected nucleoside A (0.12 g, 0.30 mmol) was dissolved in anhydrous THF (10 ml) under argon and tBuMgCl (1.0 M in THF, 1.5 ml = 1.5 mmol) was added dropwise. Phenyl (benzoxy-E-alanyl) phosphorochloridate (0.53 g, 1.50 mmol) in anhydrous THF (2 ml) was added slowly and the mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by silica gel column chromatography (4% MeOH in DCM). The resulting compound was dissolved in H ₂ O / GNo (4 ml + 1 ml) and TFA (1 ml) was added dropwise. The mixture was stirred at room temperature for 45 minutes before adding NaHCO ₃ to neutralize the solution. Column chromatography on silica gel gave the desired product. Yield: 37 mg, 20% over 2 steps. 31P-NMR (202 MHz, CD3OD): δ 3.3, 2.6. -NMR (500 MHz, CD ₃ OD): δ 8.26, 8.21 (1H, 2 x s, H-8), 7.41-7.18 (10H, m, H-Ar), 6 , 36, 6.26 (1H, 2 x dd J = 8.4, 5.8 Hz, H-1 '), 5.31-5.28, 5.27-5.24 (1H, 2 x m , H-3 '), 5.19, 5.15 (2H, 2 x s, CH2 Ph), 4.26-4.21 (1H, m, H-4'), 3.85-3.74 (2H, m, 2 x H-5 '), 2.85-2.81, 2.69-2.62, 2.60-2.57 (2H, 3 x m, H-2'), 1 , 41, 1.37 (3H, 2 x dd, J = 7.2, 1.2 Hz, NHCHCH3). MS (ES +) m / z: 626.11 (M + Na +). C ₂₆ H ₂₈ CIN ₆ O ₇ PNa required mass m / z 626.14. Reverse phase HPLC, eluting with H2O / MeOH from 100/0 to 0/100 for 35 min, flow = 1 ml / min, λ = 254, tR = 18.9, 19.6 min.

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Anticancer activity

The compounds of the present invention were compared with cladribine and compound Y, cladribine 5'phosphoramidate.

The cell lines used included

HEL92.1.7: erythroleukemia;

HL-60: promyelocytic leukemia;

KG-1: acute myeloid leukemia;

K562: chronic myeloid leukemia;

L1210: lymphoblast cell line obtained from mice with lymphocytic leukemia;

MCF7: human breast epithelial adenocarcinoma cell lines (estrogen sensitive);

NB4: Acute promyelocytic leukemia sensitive to all-trans retinoic acid;

NB4R2: Acute promyelocytic leukemia insensitive to all-trans retinoic acid;

RL: non-Hodgkin's lymphoma;

U937: histiocytic lymphoma;

Z-138: mantle cell lymphoma.

Example 1. In vitro cytotoxic studies.

Cladribine phosphoramidate derivatives were evaluated against leukemia cell lines (KG1, U937, K562, NB4R2, NB4 and HL-60) in vitro. Determined the inhibitory concentration (IC50) at which 50% of the cells are no longer viable (calculated using the MTS assay). The cells were treated with cladribine and its 3'-phosphoramidate derivatives at concentrations of 100-0.02 μM by serial dilutions and incubated for 72 h at 37 ° C, 5% CO2 in a final volume of 90 μl. Twenty microliters of MTS reagent (Promega UK Ltd, Southampton, Hants) was added to the tumor cell cultures and the reactions were incubated for the next 4 hours at 37 ° C, 5% CO2. The absorbance of the reaction after this time was recorded by spectrophotometry at 490 nm and the percentage of viable cells was calculated relative to untreated control cells in the same assay.

Tab. 1 shows in vitro results for compounds of the present invention relative to leukemia cell lines KG1, U937, K562, NB4R2, NB4 and HL-60.

Table 1

Compound KG1 μM U937 μM K562 μM NB4R2 μM NB4 μM HL-60 μM Cladribine 0, 06 0.02 0, 53 0, 06 0.04 0, 09 Y 0, 67 0, 08 8, 05 0, 63 0.23 1.75 19 0, 38 0.03 > 10 0, 11 0.24 0, 10 eighteen 0, 31 0.01 5, 14 1.29 0.07 0, 39 one 1, 76 3.06 > 10 2, 52 0.74 2, 68 15 fifty 0,4 > 10 9, 0 6, 0 twenty 2 twenty 6, 0 > 10 > 10 > 10 > 10 ten - - - - > 10 5, 3 b - - - - ten 0, 3 16 7, 0 2.0 > 10 > 10 9, 0 8, 0 17 - - - - 5, 5 3, 2 3 > 10 7.52 > 10 > 10 7, 82 > 10 4 > 10 > 10 > 10 5, 56 8, 04 > 10 eleven - - - - > 10 0, 3 14 twenty 0, 4 > 10 twenty 0, 8 thirty 5 5, 04 > 10 > 10 1, 57 2.56 4, 45 12 4, 0 2.0 ten twenty ten twenty 13 1 / o ten 7, 0 0, 8 0.3 1 / o

As can be seen, all of the compound exhibited some anti-cancer activity. It should be noted separately that Compound 19 was more active than Compound Y with respect to 4 of 6 tested cell lines, 5 'ProTide, containing the same phosphoramidate fragment as Compound 19.

Example 2. Additional in vitro cytotoxic studies.

A subset of the compounds of the present invention were then assayed for their cytotoxic activity in a wider range of different solid tumors and hematologic malignancies using the following assay.

Analysis of solid tumors and hematological malignant tumors

An in vitro viability assay was performed to assess the effects of compounds on cell viability in selected cell lines for 72 hours using the CellTiterGlo (CTG, PromegaG7573) assay. Tests were performed in duplicate with compound treatment at 9 points, 3.16 fold titration in 96-well plates for ~ 72 hours. Compound concentrations were initially 198 mM. A cell viability assay was performed using CellTiterGlo in a 96 well plate. Compounds were dissolved at 40 mM in thawed 100% DMSO. Compounds were serially diluted 3.16-fold in thawed DMSO and heated to 37 ° C before dissolving in medium (2 μl + 200 μl). After dissolving the compounds in the medium, the medium containing the compounds was heated to 37 ° C. in an incubator and then the compounds in the medium were added to the cell plates (50 μl + 50 μl) in duplicate. Final compound concentrations ranged from 198 μM to 19.9 nM. The solubility of all compounds was checked and recorded again, then the plates were immediately transferred to a CO ₂ tumor culture incubator and incubated for 3 days. The final concentration of DMSO was 0.5%.

The results of additional screening are presented in table. 2.

table 2

HL-60 promyelocytic leukemia KG-1 Spicy myelogenous leukemia Z-138 mantle cell lymphoma zones HEL92.1.7 erythroleukemia RL non-Jogkin lymphoma MCF-7 adenocarcinoma epithelium breast connected not ECso (μM ) Ab Maximum inhibition ECso (mk M) ab Maximum inhibition ECAo (mk M) ab Maximum inhibition ECso (mk M) ab Maximum inhibition B) ECAo (mk M) ab Maximum inhibition (%) ECAo (mk M) ab Maximum inhibition (B Cladribe n <0.019 85 0.17 93, 5 <0.019 99, 7 <0.019 97.4 <0.019 94.7 > 198 36, 3 6 0.23 96 1,2 98, 6 1.5 101 0.07 98.6 0.29 98.6 6, 8 102 7 0.27 98 1.5 98, 8 1.07 101 0.07 98.6 0.5 one hundred 5.45 103 8 0.46 96 2.75 98.4 3.5 one hundred 0.18 98.3 0.85 98.1 7, 97 101 nine 0, 13 87.7 0.8 96, 9 0.051 99, 9 0.044 97.2 0, 14 89.9 21 one hundred ten 0, 12 87.7 0.6 95 0.047 one hundred 0.042 97.2 0.15 89.8 22 99.2

Tab. Figure 2 shows that the compounds of the present invention are particularly effective in solid tumors as can be seen from the results for the MCF-7 cell line.

Example 3. Evaluation of cytotoxicity and activity against cancer stem cells.

An additional comparative analysis of the toxicity of compounds in the KG1a cell line over an extended dose range was performed, and the relative effect was evaluated for compounds on the leukemic stem cell compartment in the KG1a cell line over the entire dose range. Thus, experimental tests were carried out on certain compounds of the present invention, evaluating their ability to target cancer stem cells in a leukemia cell line. An acute myelocytic leukemia (AML) cell line, KG1a, was used to assess the relative effect of the compounds on the stem cell compartment. The KG1a cell line was chosen because it had a small compartment of stem cells with an excellent immunophenotype (Lin ^- / CD34 ⁺ / CD38 ^- / CD123 ⁺ ).

Materials and methods

Conditions for the KG1a cell line

KG1a cells of the cell line were maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin and 20% fetal calf serum. Then the cells were selected (105 cells / 100 μl) in 96-well plates and kept at 37 ° C in a humidified atmosphere of 5% carbon dioxide for 72 h in the presence of nucleoside analogs and their corresponding phosphoramidates at concentrations that were determined experimentally for each series of compounds ... In addition, control cultures were obtained to which no drug was added. Then, cells were harvested by centrifugation and analyzed by flow cytometry using Annexin V analysis.

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Measurement of apoptosis in vitro

The cultured cells were collected by centrifugation and then resuspended in 195 μl of calcium-enriched buffer. Then 5 μl Annexin V (Caltag Medsystems, Botolph Claydon, UK) was added to the cell suspension and the cells were kept in the dark for 10 min before washing. Finally, the cells were resuspended in 190 μl of calcium-enriched buffer along with 10 μl of propidium iodide. Apoptosis was assessed by two-color immunofluorescence flow cytometry as previously described. Then, LD ₅₀ values (the dose required to kill 50% of the cells in culture) were calculated for each nucleoside analog and phosphoramidate.

Immunophenotypic identification of the leukemic stem cell compartment KG1a cells were grown for 72 h in the presence of a wide range of concentrations of each analyte. The cells were then harvested and labeled with an antidifferentiation antibody cocktail (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE), and anti-CD123 (PERCP cy5). Then, a subpopulation expressing the leukemic stem cell (LSC) phenotype was detected and expressed as the percentage of all viable cells remaining in culture. The percentage of stem cells remaining was then plotted on a dose-response graph and the effects of the compounds were compared to 8-chloroadenosine.

Statistical analysis

The data obtained in these experiments were evaluated using one-way analysis of variance. All data were validated as a Gaussian or Gaussian approximation using the K2 omnibus criterion. LD ₅₀ values were calculated by non-linear regression and line analysis with optimization of sigmoidal dose-response curves. All statistical analyzes were performed using Graphpad Prism 6.0 software (Graphpad Software Inc., San Diego, CA).

results

In vitro cytotoxicity

In vitro drug sensitivity was assessed using the Annexin V / propidium iodide assay. The calculated LD50 values are also shown in table. 3.

Table 3

Compound LDso μM Stem cells! control: 4% Cladribine 0.18 6 eighteen 0.27 4 19 0.83 3, 1 Y 1.7 2

Both compounds 18 and 19 showed selectivity for stem cells. Compound 19 was more potent than Compound Y, 5'-ProTide containing the same phosphoramidate moiety as Compound 19. Compound 18 showed a preferential LSC targeting over cladribine.

Example 4. In vitro studies of cytotoxicity in cells infected with mycoplasma.

Tumor cell cultures were challenged with M. hyorhinis (ATCC 17981) and after two or more passages (to avoid primary inoculum error), successful infection was confirmed using the MycoAlert ™ Mycoplasma Detection Kit (Lonza, Basel, Switzerland). Although this analysis is only semi-quantitative, the maximum infection was observed 3-4 days after subculture of cells exposed to mycoplasma. Lines of permanently infected tumor cells are additionally called Cell line.Hyor. All tumor cell cultures were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany), 10 mm HEPES and 1 mm sodium pyruvate (Invitrogen). The cells were grown at 37 ° C in a humidified gas phase incubator containing 5% CO ₂ .

The cytostatic activity of the test compounds was investigated in cell lines infected and not infected with mycoplasma. When analyzing the effect of infection with M. hyorhinis, a monolayer of MCF7 and MCF7.Hyor cells were plated in 48-well microtiter plates (Nunc ™, Roskilde, Denmark) at 10,000 cells / well (Corning Inc., Corning, NY) at 100,000 cells / well. After 24 hours, cells were exposed to various concentrations of test compound and allowed to proliferate for 72 hours (to ensure sufficient cell proliferation and mycoplasma growth), after which the cells were trypsinized and counted using a Coulter counter (Analis, Suarlee, Belgium). Cell suspensions (L1210, L1210.Hyor, FM3A, and EM3A.Hyor) were plated into 96-well microtiter plates (Nunc) at 60,000 cells / well in the presence of various concentrations of test compound. The cells proliferated for 48 hours and were then counted using a Coulter counter. 50% Inhibitory concentration (IC ₅₀ ) was defined as the concentration of a compound required to reduce cell proliferation by 50%.

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Table 4. MCF7 cells infected with Mycoplasma hyorhnis (HYOR)

Compound MCF7 μM MCF7 / HYOR μM loss of activity Cladribine 0, 37 9, 3 25x 19 5, 2 26 5x Y 2, 1 33 16x

Compound 19 showed the least loss of activity against MCF7 cells infected with Mycoplasma hyorhinis than Compound Y, 5'-ProTide, containing the same phosphoramidate moiety as Compound 19 and cladribine.

Table 5. L1210 cells infected with Mycoplasma hyorhinis (HYOR)

CPF L1210 μM L1210 / HYOR μM loss of activity Cladribine 0, 4 thirty 8-kratio 6 ten 4 Quadruple

Compound 6 showed the smallest loss of activity against L1210 cells infected with Mycoplasma hyorhinis than cladribine.

Claims (16)

CLAIM 1. A compound of formula (II) or a pharmaceutically acceptable salt thereof: (Ii) R ¹ is phenyl or naphthyl; R ^{2 is} selected from C ₁ -C ₂₄ alkyl, C ₃ -C ₇ cycloalkyl, or G-alkylenephenyl; each R ³ and R ^{4 is} independently selected from H, C -C alkyl, and C ₁ -C ₃ alkylene D ⁹ ; R ⁹ is phenyl; where any phenyl, alkyl, alkylene, cycloalkyl or naphthyl group is optionally substituted with 1-4 substituents selected from halogen, nitro, cyano, NR ^a R ^a , NR ^a S (O) 2R ^a , NR ^a C (O) R ^a , NRaCONRaRa, NRa ^ Ra, 0R ^a ; ^{SR a, SORa, SO3R a,} SO2R a, SO2NR a R a, CO2R a C (O) R a, CONRaRa, CRaRaNRaRa, _C1-C4 alkyl, ^ - ^ - alkenyl, C ₂ -C ₄ -alkynyl and C ₁ -C ₄ -haloalkyl; where R ^{a is} independently selected from H and C ₁ -C ₄ -alkyl. 2. A compound according to claim 1 wherein R ¹ is phenyl. 3. A compound according to claim 2, wherein R ¹ is unsubstituted phenyl. 4. A compound according to claim 1, wherein R ¹ is 1-naphthyl. 5. A compound according to claim 4 wherein R ¹ is unsubstituted 1-naphthyl. 6. A compound according to any one of claims 1-5, wherein R ² is C ₄ -C ₈ alkyl. 7. A compound according to claim 6, wherein R ^{2 is} selected from isobutyl, tert-butyl, n-butyl, n-pentyl, CH ₂ C (Me) _3, or n-hexyl. 8. A compound according to any one of claims 1-5, wherein R ² is C ₅ -C ₇ -cycloalkyl. 9. A compound according to claim 8, wherein R ² is unsubstituted cyclohexyl. 10. The compound according to any one of claims 1 to 5, where R ² represents CHR ¹¹ -phenyl; where R _{11 is} selected from H and C1-C4-alkyl. 11. A compound according to claim 10 wherein R ² is unsubstituted benzyl. 12. A compound according to any one of claims 1-11, wherein any of R ³ and R ⁴ is H and the other is selected from H, Me, isopropyl, isobutyl and benzyl. - 28 036409 13. A compound according to claim 12, wherein R ⁴ is H and R ^{3 is} selected from H, Me, isopropyl, isobutyl and benzyl. 14. A compound according to claim 13, wherein R ⁴ is H and R ³ is Me. 15. A compound of formula (II) selected from 2-chloro-2'-deoxyadenosine-3 '- [phenyl (ethoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [phenyl (tert -butoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxy-O-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [phenyl (benzoxy -glycinyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxy-L-leucinyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [1-naphthyl (2, 2 -dimethylpropoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [1-naphthyl (pentoxy-L-leucinyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [1 -naphthyl (cyclohexoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (cyclohexoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [phenyl (2, 2-dimethylpropoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (ethoxy-2, 2-dimethylglycinyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxy-L-phenylalanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [1-naphthyl- (benzoxy-L-phenylalanyl)] phosphate, 2-chloro-2'-deoxyadenosine- 3 '- [phenyl (benzo xi-L-valinyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (isopropoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3' - [phenyl (2- butoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3 '- [phenyl (^) - 1-phenylethoxy-L-alanyl)] phosphate, 2-chloro-2'-deoxyadenosine-3'- [1-naphthyl- (benzoxy-L-alanyl)] phosphate and 2-chloro-2'-deoxyadenosine-3 '- [phenyl (benzoxy-L-alanyl)] phosphate. 16. A pharmaceutical composition for the treatment of cancer, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.